Locomotor behavior in vertebrates requires the establishment of selective connections between motor neurons in the spinal cord and muscle targets in the periphery. A regulatory network of Hox transcription factors has been directly linked with two critical steps in motor differentiation: the establishment of columnar identities which directs motor axons toward a specific target field;and the diversification of neurons within a column into motor pools, each pool targeting a single muscle. The molecular mechanisms by which Hox proteins contribute to motor neuron columnar and pool identities are not known. We have found that the forkhead homeodomain transcription factor FoxP1 is selectively expressed by two Hox-sensitive motor neuron columnar subtypes, the lateral motor column (LMC) and preganglionic column (PGC). The aim of this proposal is to further elucidate how the activities FoxP1 and Hox are coordinately regulated in motor neurons and to elucidate the downstream pathway that are critical in the Hox-dependent programs of motor neuron identity. The first aim of this proposal will explore the regulation of FoxP1 expression by Hox proteins and the mechanisms by which FoxP1 becomes selectively expressed in a subset of motor neuron subtypes. In this aim we will determine the influences of FoxP1 protein levels on the establishment of motor neuron columnar identities through overexpression of FoxP1 in vivo. In the second aim the impact of loss Foxp1 on motor neuron identity and connectivity with muscle targets will be examined. We will use anatomical and histological assays to examine the role of Foxp1 in establishing the initial patterns of motor axon projections in the limb and in defining the selection of synaptic targets. In the third aim biochemical interactions of FoxP1 and Hox proteins in the control of motor neuron-specific gene expression will be explored. The hypothesis that FoxP1 interacts directly with most or all of genes expressed in LMC motor neurons and in specific pools will be examined using chromatin immunoprecipitation assays. We will then examine the consequences of interactions between FoxP1 and Hox proteins using in vitro and in vivo assays. Together, these studies should help to provide a better understanding of how motor neuron diversity is generated and provide some of the basic insights into the mechanisms that determine the synaptic specificity of neurons in other regions of the nervous system. PUBLIC HEALTH RELEVANCE: One of the major challenges in the neural sciences is to understand how specific connections are made between neurons and their synaptic targets. The overall goal of this proposal is to elucidate the developmental programs that define the ability of motor neurons in the spinal cord to make very selective connections with muscle targets. Understanding the steps that determine the intrinsic properties of motor neurons may be essential in designing treatment strategies after spinal cord injury.